This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Defining the mechanisms governing myogenesis has advanced greatly in recent years. And yet, skeletal-muscle differentiation is a multiple step process controlled spatio-temporally by various factors at different transcriptional level. Many factors involved in myogenic process still remain mysterious. To completely explore factors involved in myogenesis, stable isotope labeling with amino acids in cell culture (SILAC), coupled with high accuracy mass spectrometry (LTQ-Orbitrap), was applied. By comparing the protein profiles of L6 myoblasts and terminal differentiated multinucleated myotubes, 894 proteins were quantified and 287 proteins changed significantly in fully differentiated myotubes in contrast to myoblasts. These differentially expressional proteins are mainly involved in inter-or intracellular signaling, protein synthesis and degradation, molecular chaperone,cell adhesion and extracelluar matrix,cell structure and motility, metabolism,substance transportation,etc. Some up-regulated proteins were found to be involved in promoting skeletal muscle differentiation for the first time, such as prohibitin-2, nestin and transcriptional activator protein pur-beta. These findings can provide new clues for understanding the mechanism of myogenesis.